water Article Bio-Removal of Methylene Blue from Aqueous Solution by Galactomyces geotrichum KL20A Margarita Contreras 1, Carlos David Grande-Tovar 1,* , William Vallejo 1 and Clemencia Chaves-López 2 1 Grupo de Fotoquímica y Fotobiología, Universidad del Atlántico, Puerto Colombia 81007, Colombia; [email protected] (M.C.); [email protected] (W.V.) 2 Faculty of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, Via R. Balzarini 1, 64100 Teramo, Italy; [email protected] * Correspondence: [email protected]; Tel.: +57-5-3599484 Received: 23 October 2018; Accepted: 7 January 2019; Published: 6 February 2019 Abstract: The conventional treatments used to remove dyes produced as a result of different industrial activities are not completely effective. At times, some toxic by-products are generated, affecting aquatic ecosystems. In this article, an efficient use of microorganisms is presented as a biodegradation technique that is a safe environmental alternative for the benefit of aquatic life. A strain of the yeast Galactomyces geotrichum KL20A isolated from Kumis (a Colombian natural fermented milk) was used for Methylene Blue (MB) bioremoval. Two parameters of the bioremediation process were studied at three different levels: initial dye concentration and growth temperature. The maximum time of MB exposure to the yeast was 48 h. Finally, a pseudo-first-order model was used to simulate the kinetics of the process. The removal percentages of MB, by action of G. geotrichum KL20A were greater than 70% under the best operating conditions and in addition, the kinetic simulation of the experimental results indicated that the constant rate of the process was 2.2 × 10-2 h−1 with a half time for biotransformation of 31.2 h. The cytotoxicity test based on the hemolytic reaction indicated that by-products obtained after the bioremoval process reached a much lower percentage of hemolysis (22%) compared to the hemolytic activity of the negative control (100%). All of these results suggest that the strain has the capacity to remove significant amounts of MB from wastewater effluents. Keywords: bioremediation; dyes; Galactomyces geotrichum; methylene blue 1. Introduction Most of the industrial effluents in developed countries have a high load of xenobiotic compounds, such as dyes. These compounds are recalcitrant, difficult to biodegrade, and toxic to aquatic species, animals, and human beings. Most of the dyes used in the textile industry are stable, resistant to biological, physical, and chemical treatments, implying that their complete removal is difficult. Due to their high solubility in water, they cause contamination in local aquatic systems when they are discharged directly without prior treatment [1]. In particular, Azo dyes are the largest synthetic chemical class of dyes that are characterized by the presence of one or more azo bonds (-N=N-). They are used to a great degree in textile, pharmaceutical, leather, food, cosmetic, painting, and large-scale printing industries due to their ease of synthesis and stability [2]. MB is an aromatic heterocyclic used in different applications (e.g., medicine [3], biological [4], and textile industries [5]). Currently, MB dye is one of the most commonly used substances for dyeing cotton, wood, and silk [6]. The dye is commonly found in industrial wastewater, which causes devastating effects on the environment [7]. Given the characteristics of solubility and stability of azo dyes, traditional methods of flocculation, sedimentation, or adsorption are not efficient in the removal of these compounds because they only Water 2019, 11, 282; doi:10.3390/w11020282 www.mdpi.com/journal/water Water 2019, 11, 282 2 of 13 transfer matter between different phases without effective degradation of the compounds [8]. Currently, such physicochemical methods are the most important ones used for the treatment of wastewater produced by the textile industry, resulting in effluents of acceptable quality, while at the same time generating waste products with high levels of toxicity. For this reason, research on the application of biological processes as a method of dye degradation has been intensified in recent years due to the resulting low toxicity. Their high efficiency [9,10], which is due in part to the inherent capacity of the microorganisms to break down organic pollutants by using them as a source of carbon and energy, or by co-metabolism [11,12], has also contributed to intensified research. The use of yeasts for the bioremoval of toxic compounds has many advantages in comparison to filamentous bacteria and fungi, since they grow rapidly and have the capacity to adapt and resist unfavorable environments [13]. The Geotrichum spp. is a filamentous fungus (Phylum Ascomycota, Order Saccharomycetales) that has transparent spores by segmentation of vegetative filaments [14]. G. geotrichum is a holomorphic organism, which means that it presents two forms of reproduction (sexual and asexual): the asexual form of multiplication gives rise to the anamorph (Geotrichum candidum) or imperfect state, while the sexual form gives rise to the teleomorph (G. geotrichum) or perfect state. These fungal species are capable of alternating a unicellular phase (yeast growth) with another mycelium (hyphae) in response to changes in various environmental factors (nutrients, CO2 pressure, pH changes, temperature). They are called “dimorphic fungi” and tend to proliferate as yeasts in tissues. However, they assume filamentous forms at room temperature in the environment [15]. This fungus is present in diverse habitats. It is found as a component of the natural flora in milk and is used as a ripening agent for cheese. Furthermore, it presents colonies of fast growth (four days of maturation), and of unlimited size. G. geotrichum is white and has a moist, yeast-like, appearance, and is easy to obtain. This yeast is characterized by true hyphae, thick (macrosiphonated) forming numerous arthroconidia, hyaline, and rectangular. The absence of blastoconidia differentiates it from the genus Trichosporon spp. [16]. Their lipases and proteases produce fatty acids and peptides that can be metabolized by other microbial populations, promoting the development of different aromas [17]. G. geotrichum can be considered one of the fungi with a large number of biotechnology applications. In fact, 38% of the publications which refer to this organism mention its capacity to biodegrade toxic compounds and its application in bioremediation processes in the treatment of wastewater for the improvement of the environment [18]. For G. geotrichum MTCC (registered at Microbial Type Culture Collection and Gene Bank, MTCC) a 100% efficiency in methyl red discoloration was reported after one hour at 30 ◦C. The composition of malt yeast medium used for decolorization studies was malt extract (3.0 g/L), yeast extract (3.0 g/L), peptone (5.0 g/L), and glucose (10.0 g/L) (pH 7.0) [19], while Waghmode et al. reported the decolorization of the azo dye Rubine by G. geotrichum MTCC 1360 within 96 h from exposure at 30 ◦C and pH 7.0 [20]. Govindwar et al. also reported a reduction of 86% via discoloration of the RY-84A dye at 50 mg L−1 after 30 h of exposure [21]. Despite extensive studies on different microorganisms for the development of biotechnological applications, this is the first time that removal of MB with G. geotrichum KL20A has been reported. In this work, the removal of MB dye with G. geotrichum KL20A was studied in order to search for alternative methods of water treatment that would be environmentally friendly and safe. 2. Materials and Methods 2.1. Yeast Isolation and Culture The yeast G. geotrichum KL20A was isolated from traditional Kumis samples collected in Valle del Cauca (in the southwest of Colombia) as reported by Chaves-López et al. [22]. The identification was carried out by sequencing of the D1/D2 region of the 26S rRNA gene and by random amplification of polymorphic DNA (RAPD-PCR). Subsequently, it was sequenced and compared with the data available in The European Molecular Biology Laboratory (EMBL) nucleotide sequence database. The strain was maintained in Petri dishes containing 12 mL of YPD-agar medium (10 g/L yeast extract, Water 2019, 11, 282 3 of 13 20 g/L dextrose, 20 g/L peptone, 20 g/L agar) and periodically reactivated for 24 h at 30 ◦C in the same growth medium [19,21]. A suspension cell solution was then prepared in YPD-broth (without agar) and a chamber count was performed using a Neubauer chamber to control the concentration of the yeast (approximately 3.2 × 107 cel/mL) to be used in the removal tests. The Neubauer chamber remains the most common method for cell counting around the world, also known as hemocytometer. 2.2. Removal Tests 2.2.1. Effects of the Methylene Blue Concentration and Temperature Methylene Blue dye (MB CI 52015 from Merck KGa, Darmstadt, Germany) was used to prepare a stock solution of 3.13 M (1000 ppm, pH 7.3) by dissolving the required amount of the dye in sterile distilled water. Concentrations of 50, 100, and 200 ppm were used for the removal tests. To this end, sterilized tubes containing 1 mL of YPD-broth medium (pH 7.0) were added to the appropriate MB concentrations (50 ppm, 100 ppm, and 200 ppm) and inoculated with 3.2 × 107 cells/mL of G. geotrichum KL20A. Decolorization of MB was studied at 25 ◦C, 30 ◦C and 35 ◦C for 48 h. Periodically, 160 µL of the samples were transferred into a 96 well microplate and the absorbance at 660 nm was read using Elisa SPR-960 reader [23]. The removal tests were carried out in triplicate with their respective targets, under the strictest biosecurity conditions to preserve the asepsis of the experiments. 2.2.2. Effects of the pH In order to study the effect of the pH on the discoloration of the MB, we performed the experiment using the optimal conditions of concentration and temperature to degrade MB (50 ppm, 35 ◦C).